Methods for Treatment of Fabry Disease

ABSTRACT

Provided are in vitro and in vivo methods for determining whether a patient with Fabry disease will respond to treatment with a specific pharmacological chaperone.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication Ser. No. 60/801,089, filed on May 16, 2006, and from U.S.provisional patent application Ser. No. 60/853,631, filed on Oct. 23,2006, each of which is incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The present invention provides methods to determine whether a patientwith Fabry disease will benefit from treatment with a specificpharmacological chaperone. The present invention exemplifies twomethods, one in vitro and one in vivo, for determining α-galactosidase Aresponsiveness to a pharmacological chaperone such as1-deoxygalactonojirimycin in patient cells. The invention also providesa method for diagnosing Fabry disease in patients suspected of havingFabry disease.

BACKGROUND

Fabry disease is a glycosphingolipid (GSL) lysosomal storage disorderresulting from an X-linked inherited deficiency of lysosomalα-galactosidase A (α-GAL), an enzyme responsible for the hydrolysis ofterminal α-galactosyl residues from glycosphingolipids (Brady et al. NEngl J Med. 1967; 276: 1163-7). A deficiency in the enzyme activityresults in a progressive deposition of neutral glycosphingolipids,predominantly globotriaosylceramide (ceramide trihexoside, CTH, GL-3),in cell of Fabry patients. Symptoms can be severe and debilitating,including kidney failure and increased risk of heart attack and stroke.Certain of the mutations cause changes in the amino acid sequence ofα-GAL that may result in the production of α-GAL with reduced stabilitythat does not fold into its correct three-dimensional shape. Althoughα-GAL produced in patient cells often retains the potential for somelevel of biological activity, the cell's quality control mechanismsrecognize and retain misfolded α-GAL in the endoplasmic reticulum, orER, until it is ultimately moved to another part of the cell fordegradation and elimination. Consequently, little or no α-GAL moves tothe lysosome, where it normally hydrolyzes GL-3. This leads toaccumulation of GL-3 in cells, which is believed to be the cause of thesymptoms of Fabry disease. In addition, accumulation of the misfoldedα-GAL enzyme in the ER may lead to stress on cells and inflammatory-likeresponses, which may contribute to cellular dysfunction and disease.

Fabry disease is classified by clinical manifestations into threegroups: a classic form with generalized vasculopathy, an atypicalvariant form with clinical manifestations limited to cardiac tissue, andlater-onset disease, which includes female carriers with mild to severeforms of the disease.

The frequency of the classical form of disease is estimated to be about1:40,000 to 1:60,000 in males, and is reported throughout the worldwithin different ethnic groups. Classically affected males have littleor no detectable α-GAL levels and are the most severely affected. Theclinical manifestations include angiokeratoma (small, raisedreddish-purple blemishes on the skin), acroparesthesias (burning inhands and feet), hypohidrosis (decreased ability to sweat), andcharacteristic corneal and lenticular opacities (The Metabolic andMolecular Bases of Inherited Disease, 8th Edition 2001, Scriver et al.,ed., pp. 3733-3774, McGraw-Hill, New York). Lipid storage may lead toimpaired arterial circulation and increased risk of heart attack orstroke. The heart may also become enlarged and the kidneys may becomeprogressively involved. Other symptoms include fever andgastrointestinal difficulties, particularly after eating. The affectedmale's life expectancy is reduced, and death usually occurs in thefourth or fifth decade as a result of vascular disease of the heart,brain, and/or kidneys.

Individuals with later-onset Fabry disease can be male or female.Late-onset Fabry disease presents as the atypical variant form, andgrowing evidence indicates there may be a significant number of“atypical variants” which are unaccounted for in the world. Females, whoinherit an X chromosome containing an α-GAL mutation, may exhibitsymptoms later in life, significantly increasing the prevalence of thisdisease. These patients typically first experience disease symptoms inadulthood, and often have disease symptoms focused on a single organ.For example, many males and females with later-onset Fabry disease haveenlargement of the left ventricle of the heart. Later-onset Fabrydisease may also present in the form of strokes of unknown cause. As thepatients advance in age, the cardiac complications of the diseaseprogress, and can lead to death.

In contrast, patients with the milder “cardiac variant” of Fabrydiseasenormally have 5-15% of normal α-GAL activity, and present withleft ventricular hypertrophy or a cardiomyopathy. These cardiac variantpatients remain essentially asymptomatic when their classically affectedcounterparts are severely compromised. Cardiac variants were found in11% of adult male patients with unexplained left ventricularhypertrophic cardiomyopathy, suggesting that Fabry disease may be morefrequent than previously estimated (Nakao et al., N. Engl. J. Med. 1995;333: 288-293).

The α-GAL gene has been mapped to Xq22 (Bishop et al., Am. J Hum. Genet.1985; 37: A144), and the full-length cDNA and entire 12-kb genomicsequences encoding α-GAL have been reported (Calhoun et al., Proc. Natl.Acad. Sci. USA. 1985; 82: 7364-7368; Bishop et al., Proc. Natl. Acad.Sci. USA. 1986; 83: 4859-4863; Tsuji et al., Eur. J. Biochem. 1987; 165:275-280; and Kornreich et al., Nucleic Acids Res. 1989; 17: 3301-3302).There is a marked genetic heterogeneity of mutations that cause Fabrydisease (The Metabolic and Molecular Bases of Inherited Disease, 8thEdition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York;Eng et al., Am. J Hum. Genet. 1993; 53: 1186-1197; Eng et al., Mol. Med.1997; 3: 174-182; and Davies et al., Eur. J. Hum. Genet. 1996; 4:219-224). To date, a variety of missense, nonsense, and splicingmutations, in addition to small deletions and insertions, and largergene rearrangements, have been reported, although the majority ofmutations are missense mutations.

Fabry disease is heterogeneous and it is often difficult to correlategenotype with phenotype. People with the same genotype often exhibitdifferent clinical symptoms and disease pathology. However, thereappears to be a correlation between residual enzyme activity and diseaseseverity, with the lower the α-GAL activity resulting in the greatestseverity of disease. Although the vast majority of α-GAL mutations aremissense mutations, with most being outside the catalytic site, itdifficult to predict which mutations result in an unstable enzyme thatcould be “rescued” by a specific pharmacological chaperone (SPC) whichstabilizes the enzyme, and which ones cannot be stabilized using a SPC.

Diagnosis of Fabry Disease

Because Fabry disease is rare, involves multiple organs, has a wide agerange of onset, and is heterogeneous, proper diagnosis is a challenge.Awareness is low among health care professionals and misdiagnoses arefrequent. Some examples of diagnoses seriously considered in patientswho were eventually diagnosed with Fabry's disease include: mitral valveprolapse, glomerulonephritis, idiopathic proteinuria, systemic lupuserythematosus, Whipple's disease, acute abdomen, ulcerative colitis,acute intermittent porphyrias, and occult malignancies. Thus, even forclassically affected males, diagnosis typically takes from about 5-7years or even longer. This is a concern because the longer a person hasFabry disease, the more damage is likely to occur in the affected organsand tissues and the more serious the person's condition may become.Diagnosis of Fabry disease is most often confirmed on the basis ofdecreased α-GAL activity in plasma or peripheral leukocytes (WBCs) oncea patient is symptomatic, coupled with mutational analysis. In females,diagnosis is even more challenging since the enzymatic identification ofcarrier females is less reliable due to random X-chromosomalinactivation in some cells of carriers. For example, some obligatecarriers (daughters of classically affected males) have α-GAL enzymeactivities ranging from normal to very low activities. Since carrierscan have normal α-GAL enzyme activity in leukocytes, only theidentification of an α-GAL mutation by genetic testing provides precisecarrier identification and/or diagnosis.

Treatment of Fabry Disease

The only approved therapy for treating Fabry disease diseases is enzymereplacement therapy, which typically involves intravenous, infusion of apurified form of the corresponding wild-type protein (Fabrazyme®,Genzyme Corp.). One of the main complications with protein replacementtherapy is attainment and maintenance of therapeutically effectiveamounts of protein in vivo due to rapid degradation of the infusedprotein. The current approach to overcome this problem is to performnumerous costly high dose infusions.

Protein replacement therapy has several additional caveats, such asdifficulties with large-scale generation, purification, and storage ofproperly folded protein; obtaining glycosylated native protein;generation of an anti-protein immune response; and inability of proteinto cross the blood-brain barrier to mitigate central nervous systempathologies (i.e., low bioavailability). In addition, replacement enzymecannot penetrate the heart or kidney in sufficient amounts to reducesubstrate accumulation in the renal podocytes or cardiac myocytes, whichfigure prominently in Fabry pathology.

Gene therapy using recombinant vectors containing nucleic acid sequencesthat encode a functional protein, or using genetically modified humancells that express a functional protein, is also being evaluated totreat protein deficiencies and other disorders that benefit from proteinreplacement. Although promising, this approach is also limited bytechnical difficulties such as the inability of vectors to infect ortransduce dividing cells, low expression of the target gene, andregulation of expression once the gene is delivered.

A third, relatively recent approach to treating some enzyme deficienciesinvolves the use of small molecule inhibitors to reduce production ofthe natural substrate of deficient enzyme proteins, thereby amelioratingthe pathology. This “substrate reduction” approach has been specificallydescribed for a class of about 40 related enzyme disorders calledlysosomal storage disorders that include glycosphingolipid storagedisorders. The small molecule inhibitors proposed for use as therapy arespecific for inhibiting the enzymes involved in synthesis ofglycolipids, reducing the amount of cellular glycolipid that needs to bebroken down by the deficient enzyme. This approach is also limited inthat glycolipids are necessary for biological function, especiallyneurological function, and excessive deprivation may cause adverseeffects.

It has previously been shown that the binding of small moleculeinhibitors of enzymes associated with LSDs can increase the stability ofboth mutant enzyme and the corresponding wild-type enzyme (see U.S. Pat.Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829, and7,141,582 all incorporated herein by reference). In particular, it wasdiscovered that administration of small molecule derivatives of glucoseand galactose, which are specific, selective competitive inhibitors forseveral target lysosomal enzymes, effectively increased the stability ofthe enzymes in cells in vitro and, thus, increased trafficking of theenzymes to the lysosome. Thus, by increasing the amount of enzyme in thelysosome, hydrolysis of the enzyme substrates is expected to increase.The original theory behind this strategy was as follows: since themutant enzyme protein is unstable in the ER (Ishii et al., Biochem.Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is retardedin the normal transport pathway (ER→Golgi apparatus→endosomes→lysosome)and prematurely degraded. Therefore, a compound which binds to andincreases the stability of a mutant enzyme, may serve as a “chaperone”for the enzyme and increase the amount that can exit the ER and move tothe lysosomes. In addition, because the folding and trafficking of somewild-type proteins is incomplete, with up to 70% of some wild-typeproteins being degraded in some instances prior to reaching their finalcellular location, the chaperones can be used to stabilize wild-typeenzymes and increase the amount of enzyme which can exit the ER and betrafficked to lysosomes. This strategy has been shown to increaseseveral lysosomal enzymes in vitro and in vivo, includingβ-glucocerebrosidase and α-glucosidase, deficiencies of which areassociated with Gaucher and Pompe disease, respectively.

However, as indicated above, successful candidates for SPC therapy musthave a mutation which results in the production of an enzyme that hasthe potential to be stabilized and folded into a conformation thatpermits trafficking out of the ER. Mutations which severely truncate theenzyme, such as nonsense mutations, or mutations in the catalytic domainwhich prevent binding of the chaperone, will not likely be “rescuable”or “enhanceable” using SPC therapy. While missense mutations outside thecatalytic site are more likely to be rescuable using SPCs, there is noguarantee, necessitating screening for responsive mutations. This meansthat, even when Fabry disease is diagnosed by detecting deficient α-GALactivity in WBCs, it is very difficult, if not impossible, to predictwhether a particular Fabry patient will respond to treatment with anSPC. Moreover, since WBCs only survive for a short period of time inculture (in vitro), screening for SPC enhancement of α-GAL is difficult.

In order to apply SPC therapy effectively, a broadly applicable, fastand efficient method for screening patients for responsiveness to SPCtherapy needs to be adopted prior to initiation of treatment. Thus,there remains in the art a need for relatively non-invasive methods torapidly assess enzyme enhancement with potential therapies prior tomaking treatment decisions, for both cost and emotional benefits to thepatient.

SUMMARY OF THE INVENTION

The present invention provides two methods for determining whether apatient will be a candidate for SPC therapy. Specifically, the presentinvention provides in vitro and in vivo assays to evaluate α-GALactivity in blood cells derived from patients with Fabry disease in thepresence or absence of an SPC. The present invention also includes thebasis for evaluation of SPC as a treatment option for any number ofother protein abnormalities and/or enzyme deficiencies. The presentinvention also provides for diagnostic kits containing the componentsrequired to perform the assay. The present invention further provides animproved method of diagnosing Fabry disease by determining α-GALactivity in T cells from patients suspected of having Fabry disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Time course for enhancement for A97V. T-cells bearing the A97Vmutation in α-Gal A were cultured in the absence (open circles) orpresence (filled circles) of 20 μM DGJ for one to four days then assayedfor α-GAL activity. Changing media after 2 days and replacing with freshmedia with (open triangle) or without DGJ (filled triangle) had noeffect on the observed enzyme activity.

FIG. 2A-C. Concentration Dependence of DGJ in T-cells from normalcontrol and Fabry patients. T-cells from normal individuals (2A) wereincubated for 3 days with DGJ from 2 nM to 200 μM then assayed for α-GALactivity. The results of three experiments on different days are shown.T cells from Fabry patients with the A97V (2B), R112H (2C), or R301Q(2C) mutations, respectively, were cultured with DGJ from 2 nM to 200 μMthen assayed for α-GAL activity. Three independent sets of DGJ dosageexperiments, each of which were perfoiined with triplicate sets areshown.

FIG. 3. T-cells from various Fabry patients were cultured in the absenceof or in the presence of 20 μM DGJ for three days then assayed for α-GALactivity. Percent of the average normal of specific activity of theα-GAL were graphed to show the effect of DGJ rescue on the differentgenotypes of Fabry patients. The lower panel shows the Western blotresults for each mutation, probed with polyclonal rabbit antibodyspecific for α-GAL. This demonstrates increased protein stability forenhanceable mutations A97V and R301Q and no increase in protein amountfor the mutations R356W, G132R and A143P.

FIG. 4. Graphical representation of in vivo enhancement of α-GALactivity in WBC of Fabry patients following treatment with DGJ.

FIG. 5. Graphical representation comparing in vitro and in vivoenhancement of α-GAL activity for 10 genotypes.

DETAILED DESCRIPTION

The present invention provides two assays to allow the accuratedetermination of whether an SPC enhances enzyme activity from cellsderived from patients with Fabry disease. These assays permit adetermination of whether the patient will be a candidate for SPCtherapy. The new in vitro assay is extremely sensitive and can beperformed on isolated T cells which do not need to be extensivelycultured and maintained in vivo, which speeds up the time required toperform the assay (as compared to when fibroblasts are used). This assayalso can be used as a diagnostic assay for patients suspected of havingFabry disease, especially females, since it is more sensitive than theWBC assay typically used for detecting α-GAL activity. The new in vivoassay is similarly non-invasive and provides a very reliable method fordetermining whether a SPC therapy will be effective in a particularpatient. In addition, in conjunction with genotyping, both assaysprovide a method for determining whether newly discovered α-GALmutations (such as spontaneous mutations) cause the α-GAL to misfoldand, thus potentially would be “rescuable” using SPCs.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each teiin is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

The term “Fabry disease” refers to an X-linked inborn error ofglycosphingolipid catabolism due to deficient lysosomal α-galactosidaseA activity. This defect causes accumulation of globotriaosylceramide(ceramide trihexoside) and related glycosphingolipids in vascularendothelial lysosomes of the heart, kidneys, skin, and other tissues.

The term “atypical Fabry disease” refers to patients with primarilycardiac manifestations of the α-GAL deficiency, namely progressiveglobotriaosylceramide (GL-3) accumulation in myocardial cells that leadsto significant enlargement of the heart, particularly the leftventricle.

A “carrier” is a female who has one X chromosome with a defective α-GALgene and one X chromosome with the normal gene and in whom X chromosomeinactivation of the normal allele is present in one or more cell types.A carrier is often afflicted with Fabry disease.

A “patient” refers to a subject who has been diagnosed with a particulardisease. The patient may be human or animal. A “Fabry disease patient”refers to an individual who has been diagnosed with Fabry disease andhas a mutated α-GAL as defined further below. Characteristic markers ofFabry disease can occur in male hemizygotes and female carriers with thesame prevalence, although females typically are less severely affected.

Human α-galactosidase A (α-GAL) refers to an enzyme encoded by the humanGla gene. The human α-GAL enzyme consists of 429 amino acids and is inGenBank Accession No. U78027.

As used herein in one embodiment, the term “mutant α-GAL” includes anα-GAL which has a mutation in the gene encoding α-GAL which results inthe inability of the enzyme to achieve a stable conformation under theconditions normally present in the ER. The failure to achieve a stableconformation results in a substantial amount of the enzyme beingdegraded, rather than being transported to the lysosome. Such a mutationis sometimes called a “conformational mutant.”

Non-limiting, exemplary α-GAL mutations associated with Fabry diseasewhich result in unstable α-GAL include L32P; N34S; T41I; M51K; E59K;E66Q; I91T; A97V; R100K; R112C; R112H; F113L; T141L; A143T; G144V;S148N; A156V; L166V; D170V; C172Y; G183D; P205T; Y207C; Y207S; N215S;A228P; S235C; D244N; P259R; N263S; N264A; G272S; S276G; Q279E; Q279K;Q279H; M284T; W287C; I289F; M296I; M296V; L300P; R301Q; V316E; N320Y;G325D; G328A; R342Q; E358A; E358K; R363C; R363H; G3705; and P409A.

As used herein, the term “specific pharmacological chaperone” (“SPC”) or“pharmacological chaperone” refers to any molecule including a smallmolecule, protein, peptide, nucleic acid, carbohydrate, etc. thatspecifically binds to a protein and has one or more of the followingeffects: (i) enhances the formation of a stable molecular conformationof the protein; (ii) induces trafficking of the protein from the ER toanother cellular location, preferably a native cellular location, i.e.,prevents ER-associated degradation of the protein; (iii) preventsaggregation of misfolded proteins; and/or (iv) restores or enhances atleast partial wild-type function and/or activity to the protein. Acompound that specifically binds to e.g., α-GAL, means that it binds toand exerts a chaperone effect on α-GAL and not a generic group ofrelated or unrelated enzymes. More specifically, this term does notrefer to endogenous chaperones, such as BiP, or to non-specific agentswhich have demonstrated non-specific chaperone activity against variousproteins, such as glycerol, DMSO or deuterated water, i.e., chemicalchaperones (see Welch et al., Cell Stress and Chaperones 1996;1(2):109-115; Welch et al., Journal of Bioenergetics and Biomembranes1997; 29(5):491-502; U.S. Pat. Nos. 5,900,360; 6,270,954; and6,541,195). In the present invention, the SPC may be a reversiblecompetitive inhibitor.

A “competitive inhibitor” of an enzyme can refer to a compound whichstructurally resembles the chemical structure and molecular geometry ofthe enzyme substrate to bind the enzyme in approximately the samelocation as the substrate. Thus, the inhibitor competes for the sameactive site as the substrate molecule, thus increasing the Km.Competitive inhibition is usually reversible if sufficient substratemolecules are available to displace the inhibitor, i.e., competitiveinhibitors can bind reversibly. Therefore, the amount of enzymeinhibition depends upon the inhibitor concentration, substrateconcentration, and the relative affinities of the inhibitor andsubstrate for the active site.

Following is a description of some specific pharmacological chaperonescontemplated by this invention:

1-deoxygalactonojirimycin refers to a compound having the followingstructures:

This term includes both the free base and any salt forms. Thehydrochloride salt of DGJ is known as migalastat hydrochloride(Migalastat).

Still other SPCs for α-GAL are described in U.S. Pat. Nos. 6,274,597,6,774,135, and 6,599,919 to Fan et al., and includeα-3,4-di-epi-homonojirimycin, 4-epi-fagomine, andα-allo-homonojirimycin, N-methyl-deoxygalactonojirimycin,β-1-C-butyl-deoxygalactonojirimycin, and α-galacto-homonojirimycin,calystegine A₃, calystegine B₂, calystegine B₃, N-methyl-calystegine A₃,N-methyl-calystegine B₂ and N-methyl-calystegine B₃.

As used herein, the term “specifically binds” refers to the interactionof a pharmacological chaperone with a protein such as α-GAL,specifically, an interaction with amino acid residues of the proteinthat directly participate in contacting the pharmacological chaperone. Apharmacological chaperone specifically binds a target protein, e.g.,α-GAL, to exert a chaperone effect on α-GAL and not a generic group ofrelated or unrelated proteins. The amino acid residues of a protein thatinteract with any given pharmacological chaperone may or may not bewithin the protein's “active site.” Specific binding can be evaluatedthrough routine binding assays or through structural studies, e.g.,co-crystallization, NMR, and the like. The active site for α-GAL is thesubstrate binding site.

“Deficient α-GAL activity” refers to α-GAL activity in cells from apatient which is below the normal range as compared (using the samemethods) to the activity in normal individuals not having or suspectedof having Fabry or any other disease (especially a blood disease).

As used herein, the terms “enhance α-GAL activity” or “increase α-GALactivity” refer to increasing the amount of α-GAL that adopts a stableconformation in a cell contacted with a pharmacological chaperonespecific for the α-GAL, relative to the amount in a cell (preferably ofthe same cell-type or the same cell, e.g., at an earlier time) notcontacted with the pharmacological chaperone specific for the α-GAL.This term also refers to increasing the trafficking of α-GAL to thelysosome in a cell contacted with a pharmacological chaperone specificfor the α-GAL, relative to the trafficking of α-GAL not contacted withthe pharmacological chaperone specific for the protein. These termsrefer to both wild-type and mutant α-GAL. In one embodiment, theincrease in the amount of α-GAL in the cell is measured by measuring thehydrolysis of an artificial substrate in lysates from cells that havebeen treated with the SPC. An increase in hydrolysis is indicative ofincreased α-GAL activity.

The term “α-GAL activity” refers to the normal physiological function ofa wild-type α-GAL in a cell. For example, α-GAL activity includeshydrolysis of GL-3.

A “responder” is an individual (diagnosed with or suspected of havingFabry disease) whose cells exhibit sufficiently increased α-GALactivity, and/or amelioration of symptoms or improvement in surrogatemarkers, in response to contact with an SPC. Non-limiting examples ofimprovements in surrogate markers for Fabry disease include increases inα-GAL levels or activity in cells (e.g., fibroblasts) and tissue;reductions in of GL-3 accumulation; decreased plasma concentrations ofhomocysteine and vascular cell adhesion molecule-1 (VCAM-1); decreasedGL-3 accumulation within myocardial cells and valvular fibrocytes;reduction in cardiac hypertrophy (especially of the left ventricle),amelioration of valvular insufficiency, and arrhythmias; amelioration ofproteinuria; decreased urinary concentrations of lipids such as CTH,lactosylceramide, ceramide, and increased urinary concentrations ofglucosylceramide and sphingomyelin (Fuller et al., Clinical Chemistry.2005; 51: 688-694); the absence of laminated inclusion bodies (Zebrabodies) in glomerular epithelial cells; improvements in renal function;mitigation of hypohidrosis; the absence of angiokeratomas; andimprovements hearing abnormalities such as high frequency sensorineuralhearing loss progressive hearing loss, sudden deafness, or tinnitus.Improvements in neurological symptoms include prevention of transientischemic attack (TIA) or stroke; and amelioration of neuropathic painmanifesting itself as acroparaesthesia (burning or tingling inextremities).

The dose that achieves one or more of the aforementioned responses is a“therapeutically effective dose.”

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, 18th Edition, or other editions.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an mRNA band on a gel, a cDNA, or a restriction fragment.In another embodiment, an isolated nucleic acid is preferably excisedfrom the chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acids include sequences inserted into plasmids,cosmids, artificial chromosomes, and the like. Thus, in a specificembodiment, a recombinant nucleic acid is an isolated nucleic acid. Anisolated protein may be associated with other proteins or nucleic acids,or both, with which it associates in the cell, or with cellularmembranes if it is a membrane-associated protein. An isolated organelle,cell, or tissue is removed from the anatomical site in which it is foundin an organism. An isolated material may be, but need not be, purified.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 10- or5-fold, and more preferably within 2-fold of a given value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Method

To easily determine whether SPC therapy will be a viable treatment forFabry patients, including female carriers, simple, non-invasive DGJrescue assay of α-GAL activity in WBCs, or subsets of WBCs, from Fabrypatients was developed.

I. In Vitro Assay

In one embodiment, the diagnostic method of the present inventioninvolves purifying T cells and establishing T cell cultures from bloodspecimens from Fabry patients (or patients suspected of having Fabrydisease). T cell cultures are then treated with or without an SPC, e.g.,DGJ, for a sufficient time period to demonstrate enhancement (i.e.,increase) of α-GAL activity. The T cells are then lysed, and the lysateis used in an assay to determine enzyme activity. A sufficient increasein α-GAL activity in the lysates from cells treated with the SPC overthe activity in the lysates from untreated cells indicates that thepatient will likely respond to SPC therapy (i.e., the patient will be a“responder”).

This embodiment can be carried out as follows.

White Blood Cell Separation

The WBCs are prepared using standard techniques, e.g., collection,centrifugation, separation, and washing. More specifically, they can beprepared according to the following steps:

-   -   1. A blood sample is drawn from a Fabry patient. In specific        embodiments, approximately 8 to 10 mL are drawn into an        appropriate container such as a CPT tube from Becton-Dickenson        (containing an anti-coagulant and a separation medium).    -   2. The blood sample is centrifuged to separate red blood cells        from white blood cells and plasma. Typically, this step can be        performed at room temperature (18-25° C.) at about 1800×g with a        tabletop centrifuge for about 20-30 minutes, or until the red        blood cells are separated from plasma and white blood cells        (WBCs). However, other known white blood cell separation        techniques may also be used, e.g., Ficoll-Hypaque, Percoll or        other similar density gradients. In an alternative embodiment, T        cells are enriched from WBCs using antibody-mediated or magnetic        separation using negative selection to remove other cell types        in order to obtain unbound T cells. Any known technique for        enriching for T cells can be used, although the more expedient,        least expensive methods are preferred.    -   3. Half of the plasma layer is discarded (without disturbing the        white blood cell layer) and remaining fluid containing white        blood cells is transferred to a centrifuge tube.    -   4. The WBCs are then pelleted and washed for two or more times        by re-suspending the pelleted cells in an appropriate isotonic        buffer, e.g., PBS, followed by centrifugation for about 15-20        minutes at about 320×g.    -   5. The pellet is then re-suspended with a small volume of        appropriate isotonic buffer, e.g., PBS. Half of the pellet is        transferred to a labeled cryovial for freezing. The other half        is used for establishing T cell cultures as described below. The        sample that is to be frozen is centrifuged and then resuspended        in a small volume of appropriate isotonic buffer, e.g., RPMI        1640 plus DMSO, prior to freezing.

T-Cell Cultures

In one embodiment, T-cell cultures are established by stimulating the Tcells present in the WBC preparation, for example, according to thefollowing procedure.

-   -   1. The washed cells from above are re-suspended in an        appropriate cell culture medium, such as RPMI supplemented with        T cell stimulatory cytokines and/or mitogens. Suggested        stimulatory cytokines include IL-2, IL-12, IL-15        phytohemagglutinin (PHA), concanavalin A (con A), and pokeweed        mitogen. In a particular embodiment, the WBCs are re-suspended        in an appropriate volume of RPMI 1640 medium supplemented with        FBS, IL-2 and a stimulatory concentration of PHA. They can then        be transferred to an appropriate culture vessel and incubated        for sufficient time to expand, e.g., about 2-3 days.    -   2. After the T cells are expanded, they may be frozen, e.g., at        about 3×10⁶ cells/vial using RPMI 1640 medium supplemented for        cryopreservation, e.g., containing FCS and DMSO. This is        sufficient to thaw 5 mL of culture at 5×10⁵ viable cells/mL.

It is noted that one of ordinary skill in the art will be able toascertain appropriate amounts of T cell stimulatory cytokines ormitogens, although typically such agents are added at amounts frombetween about 1 ng/ml and about 25 ng/ml (or about 100 U/ml) forcytokines. For mitogens, concentrations range from about 10 ng/ml toabout 10 μg/ml for mitogens with most being effective in the low μg/mlrange.

Enzyme Activity/Enhancement Assay

Typically, T cells isolated above (e.g., approximately 2.5×10⁶) aregrown in culture medium (preceded by thawing if they are frozen), in anappropriate culture vessel in the absence or presence of the SPC, e.g.,DGJ, for enough time to evaluate the change in α-GAL activity, e.g., 2or 3 days. Doses of DGJ expected to enhance α-GAL are in a range fromabout 2 nM to about 150 μM; preferably about 1 μM to 100 μM, and morepreferably about 5 μM to 50 μM. In one specific embodiment, DGJ is addedat about 20 μM. Cells can be harvested by centrifugation and washedtwice with PBS. Pellets can be stored frozen at −80° C. until assayedfor enzyme activity.

Cells are then lysed by the addition of lysis buffer (or deionizedwater) and physical disruption (pipetting, vortexing and/or agitation,and/or sonication) at room temperature or on ice, followed by pooling ofthe lysates on ice, then splitting the pooled lysate into small aliquotsand freezing.

The lysates can be thawed immediately prior to the assay and should besuspended by use of a vortex mixer and sonicated prior to addition toappropriate wells e.g., in a microplate. N-acetylgalactosamine (GalNAc)is then added to each well (to inhibit α-galactosidase B), followed by ashort incubation. 4-methylumbelliferyl-α-D-galactopyranoside (4-MU Gal),or other appropriate labeled DGJ substrate, is then added and the plateis gently mixed for a brief period of time, covered, and incubated at37° C. for a sufficient time for substrate hydrolysis, usually about 1hour. To stop the reaction, NaOH-glycine buffer, pH 10.7, is added toeach well and the plate is read on a fluorescent plate reader (e.g.Wallac 1420 Victor3™ or similar instrument). Excitation and emissionwavelengths were customarily set at 355 nm and 460 nm, respectively. Oneunit of enzyme activity is defined as the amount of enzyme thatcatalyzes the hydrolysis of 1 nmole of 4-methylumbelliferone per hour.For each patient sample at least three normal samples should be testedconcurrently.

Various modifications of this assay will be readily ascertainable to oneof ordinary skill in the art. Examples of artificial substrates that canbe used to detect α-GAL activity include but are not limited top-nitrophenyl-α-D-galactopyranoside and 4-MU GAL. Obviously, onlysubstrates that can be cleaved by human α-GAL are suitable for use. Itis noted that while use of a fluorogenic substrate is preferred, othermethods of determining α-GAL activity are contemplated for use in themethod, including using chromogenic substrates or immunoquantificationtechniques.

Diagnosis and Prognosis.

The T cell assay can be easily modified for use as a diagnostic assay todiagnose Fabry disease by simply eliminating the step of culturing the Tcells in the presence of DGJ prior to performing the enhancement assay.The activity of α-GAL in T cells established from an individualsuspected of having Fabry disease can instead be quantitated using Tcells from a normal individual as a control. Moreover, both α-GALactivity and SPC enhancement assays can be performed almostsimultaneously using the same T cells derived from one patient sample.Since T cells may express more α-GAL (α-GAL in normal T cells ascompared with WBCs is much higher), it will be easier to confirm withmore certainty whether a patient has α-GAL activity below the normalrange because the margin of error will be smaller. Accordingly, use ofthe T cell assay could potentially prevent misdiagnoses.

In addition, the modified assay also can be used to periodically monitorthe progress of patients in whom SPC therapy was initiated to confirmthat α-GAL activity remains increased relative to prior to treatmentinitiation.

II. In Vivo Assay

In a second embodiment, WBCs are evaluated for α-GAL enhancement by anSPC in vivo. In this embodiment, α-GAL activity in WBCs derived frompatients is assessed prior to SPC administration, in order to obtain abaseline value. Patients are then administered DGJ daily (e.g., 150mg/day) for a sufficient time period, e.g., about 10 days to about 2weeks, followed by extraction of blood and determination of changes inα-GAL activity from the baseline value. Culturing the dells either priorto or following administration is not required.

The dose and dosing regimen of DGJ administration during the in vivoevaluation period may vary depending on the patient since there is somuch heterogeneity among mutations, and depending on the patient'sresidual α-GAL activity. As non-limiting examples, the following dosesand regimens are expected to be sufficient to increase α-GAL in most“rescuable” individuals: 25 mg b.i.d; 50 mg once a day; 50 mg b.i.d.; 50mg once every other day, 75 mg once a day; 75 mg b.i.d.; 100 mg once aday; 100 mg b.i.d.; 150 mg once a day; 150 mg b.i.d., 150 mg once everyother day; 250 mg once a day; 250 mg b.i.d. and 250 mg once every otherday. In specific embodiments, the doses are 50 mg once a day; 50 mg onceevery other day; 150 mg once a day; 150 mg once every other day.

Administration of DGJ according to the present invention may be in aformulation suitable for any route of administration, but is preferablyadministered per os in an oral dosage form such as a tablet, capsule orsolution. As one example, the patient is orally administered capsuleseach containing 25 mg, 50 mg, 75 mg or 100 mg or combinations thereof.For this assay, in the case of oral administration, it is preferred thatthe patient be administered the DGJ without food (e.g., no food 2 hoursbefore and for 2 hours after dosing) since bioavailability may be lowerif taken with food, thereby risking inaccurate results.

Patients who have been on other therapies, such as ERT, should ceasetreatment for at least about 28 days prior to the in vivo assay toensure the most accurate results.

White Blood Cell Separation

WBCs are isolated and separated as described above for the T cell invitro assay. However, no RPMI media or DMSO is to be added to thepellets prior to freezing (as per step 5 above).

Enzyme Activity/Enhancement Assay

Pellets are thawed on ice and cells are then lysed by the addition oflysis buffer and physical disruption (such as by use of a vortex mixerand agitation, and/or sonication at room temperature) for a sufficienttime, followed by pooling of the lysates in a polypropylene tube on ice,then splitting of the pooled lysate into aliquots for freezing.

The WBC lysates are then thawed on ice and mixed (again, by sonicationand/or vortexing). Samples of each lysate, as well as standards andnegative controls, are then added to appropriate wells in e.g., a 24 or96 well microplate. Equal amounts of GalNAc are added to each well ine.g., citrate/phosphate buffer, pH 4.6, followed by addition of alabeled substrate, such as 4-MU Gal (also in citrate/phosphate buffer,pH 4.6) to all wells, and incubation for a short time at ambienttemperature. The plate is then mixed briefly and incubated at 37° C. fora sufficient time period to permit substrate hydrolysis, e.g., about 1hour. After the sufficient time period, the reaction is stopped by theaddition of stop buffer and the plate is read on a fluorescent platereader (e.g., Wallac 1420 Victor3™) to determine enzyme activity perwell.

Various modifications of this assay will be readily ascertainable to oneof ordinary skill in the art. Examples of artificial substrates that canbe used to detect α-GAL activity include but are not limited top-nitrophenyl-α-D-galactopyranoside and 4-MU Gal. Obviously, onlysubstrates that can be cleaved by human α-GAL are suitable for use. Itis noted that while use of a fluorogenic substrate is preferred, othermethods of determining α-GAL activity are contemplated for use in themethod, including using chromogenic substrates or immunoquantificationtechniques.

Eligibility Determination Criteria

The criteria for determining eligibility for SPC therapy depends on thepatient's residual enzyme activity at baseline, i.e., the activitydetermined in the untreated T cells in the in vitro assay, or theactivity in the WBCs prior to SPC administration in the in vivo assay.The lower the residual activity, the greater enhancement necessary inorder for a patient to be considered a likely responder to treatment.

In one embodiment, the criteria for determining eligibility for the invitro assay are as follows:

-   -   If residual α-Gal A activity in lymphocytes is less than 1% of        normal, then α-GAL activity after incubation with DGJ must be at        least 2% of normal;    -   If residual α-GAL activity in lymphocytes is between 1% of        normal and <3% of normal, then α-GAL activity after incubation        with DGJ must be at least 2× the baseline level;    -   If residual α-GAL activity in lymphocytes is between 3% of        normal and <10% of normal, then α-GAL activity after incubation        with DGJ must be at least 3% of normal higher than the baseline        level; and    -   If residual α-GAL activity in lymphocytes is 10% of normal or        more, then α-GAL activity after incubation with DGJ must be at        least 1.3× the baseline level.

In an alternative embodiment, patients with Fabry disease could becategorized as eligible for SPC therapy if their α-GAL activity in Tcells in the presence of an SPC such as DGJ is at least about 35-40nmol/hr/mg protein, which is about 58% of normal. According to thepresent invention, the average specific was too variable to report as aglobal mean. Accordingly patient T-cell samples were compared inactivity to at least three normal controls collected within 48 h of thecollection date for the patient specimen and grown under identicalconditions (see Example 1). As a comparison, α-GAL activity in T cellsfrom Fabry patients with the A97V, R301Q, and R111H at baseline was 8nmole/hr/mg protein, 4 nmol/hr/mg and 1.8 nmol/hr/mg. T cells expresshigher levels of α-GAL compared with other WBCs, so it is expected thatα-GAL activity in a culture enriched for T cells will be significantlyhigher than what is considered normal in total WBCs (21 nmol/h/mg ofprotein to about 50 nmol/h/mg of protein; Desnick et al., The Metabolicand Molecular Bases of Inherited Diseases. 8th Edition 2001, Scriver etal., ed., pp. 3733-3774, McGraw-Hill, New York). For a comparison, threeFabry patients having the mutations R220X, R356W, and G132R had WBCα-GAL activity of 0.22, 0.18, and 0.26 nmol/hr/mg protein, respectively.

In one embodiment, for the in vivo assay, the following criteria areused to determine eligibility criteria:

-   -   If baseline α-GAL is less than 1% of normal, then Day 15 α-GAL        activity after treatment with DGJ must be at least 2% of normal;    -   If baseline α-GAL is between 1% of normal and <5% of normal,        then α-GAL activity must be at least 2× the baseline level        following the treatment period;    -   If baseline α-GAL is between 5% of normal and <10% of normal,        then α-GAL activity must be at least 5% of normal higher than        the baseline level following the treatment period; and    -   If baseline α-GAL is 10% of normal or more, then α-GAL activity        must be at least 1.5× the baseline level following the treatment        period.

In an alternative embodiment, an increase in activity of at least about20% in the cells cultured with SPC over the activity in the cells notcultured with SPC, in either the in vitro or in vivo assay, may beindicative that the patient will have a clinically relevant(therapeutically effective) response to SPC therapy.

This discovery provides a method for improving the diagnosis of andfacilitating clinical treatment decisions for Fabry disease inparticular, and lysosomal storage disease in general. Moreover, thismethod can be extended to a wide range of genetically defined diseasesin appropriate cell types. This class of disease includes the otherlysosomal storage disorders, Cystic Fibrosis (CFTR) (respiratory orsweat gland epithelial cells), familial hypercholesterolemia (LDLreceptor; LPL-adipocytes or vascular endothelial cells), cancer (p53;PTEN-tumor cells), and amyloidoses (transthyretin) among others.

Kits

The present invention also provides for a commercial diagnostic test kitin order to make therapeutic treatment decisions. The kit provides allmaterials discussed above and more particularly in, the Examples below,for preparing and running each assay in one convenient package, with theobvious exception of patient blood, optionally including instructionsand an analytic guide.

As one non-limiting example, a kit for evaluating α-GAL activity maycontain, at a minimum:

-   -   a. at least one T cell stimulatory agent;    -   b. a specific pharmacological chaperone;    -   c. a chromogenic or fluorogenic substrate for the enzyme assay        (including an appropriate standard); and    -   d. GalNAc.        The kit may also contain instructions for optimally performing        the protein enhancement assay. In another embodiment, the kit        will contain the appropriate tubes, buffers (e.g., lysis        buffer), and microplates.

In one embodiment, the SPC is supplied in dry form, and will bere-constituted prior to addition.

In another embodiment, the invention provides a kit for the diagnosis ofFabry disease. In this embodiment, the SPC is not included in the kitand the instructions are tailored specifically to diagnosis.

Patients that test positive for enzyme enhancement with an SPC can thenbe treated with that agent, whereas patients who do not display enzymeenhancement with a specific agent can avoid treatment which will savemoney and prevent the emotional toll of not responding to a treatmentmodality.

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification. The invention is therefore to be limitedonly by the terms of the appended claims along with the full scope ofequivalents to which the claims are entitled.

Example 1: In Vitro Method for Evaluating Effects of an SPC on α-GALActivity

The present Example provides the in vitro diagnostic assay to determinea Fabry patient's responsiveness to a specific pharmacologicalchaperone.

A. Preparation of Human WBC Pellets for Growth of T Lymphocytes

1. Materials:

-   -   CPT tube: Becton-Dickenson (BD Vacutainer® CPT™ Cell Preparation        Tube with Sodium Citrate, cat #362761).    -   Human IL-2 (recombinant), PreProTECH, cat #200-02    -   Phytohemagglutinin (M Form) (PHA), liquid, Invitrogen, cat        #10576-015    -   RPMI-1640 medium, Mediatech Inc., cat #10-040-CV    -   Fetal Bovine Serum, Mediatech Inc., cat #35-010-CV    -   Citric acid, monohydrate, ACS, Mallinckrodt, cat #0627    -   Sodium phosphate dibasic (Na₂HPO₄), ACS, Mallinckrodt cat #7917    -   Sodium hydroxide, volumetric solution 10N, Mallinckrodt cat #        H385    -   Phosphoric acid, ACS, Mallinckrodt cat # PX0995-3    -   4-MU α-D-galactopyranoside (4-MU-Gal), Sigma cat # M-7633    -   N-Acetyl-D-galactosamine (GalNAc), Sigma cat # A-2795    -   4-methylumbelliferone (4-MU), Sigma cat # M-1381    -   Glycine, tissue culture grade, Fisher cat # BP381    -   Double deionized water    -   Dulbecco's Phosphate Buffered Saline, PBS, (without Ca, without        Mg), Mediatech Inc. cat #21-031-CV    -   Micro BCA Protein Assay Kit, Pierce cat #23235    -   96-well microtiter plates, Costar black polystyrene 96 well        round bottom, cat #3792    -   Costar 24-well tissue culture treated microplates, Corning Life        Sciences, cat #3526    -   15 mL polypropylene Falcon tube, Becton Dickinson, cat #352097    -   Sterile Cryovials    -   Humidified 5% CO₂, 37° C. incubator    -   37° C. water bath    -   Fluorescence plate reader

2. WBC Separation:

-   -   Patient blood was drawn into an 8 mL CPT tube, which has been        stored at 18-25° C.    -   immediately after collecting blood, it was mixed by inverting        the tube 8-10 times.    -   The tube was centrifuged at room temperature (18-25° C.) for 30        minutes at 1800× g using a tabletop centrifuge equipped with        swinging buckets. Universal precautions for handling blood        specimens were taken, including the use of a closed canister        type bucket for centrifugation.    -   Following centrifugation, several layers of the blood        composition become distinguishable which represented separation        of the red blood cells from the plasma and white cells. If this        does not occur, warm in hands for 5 minutes and centrifuge        again.

3. Washing of WBC's

-   -   Half of the plasma layer was aspirated by vacuum and discarded        without disturbing the white cell layer. All of the remaining        fluid, including the cell layer, was transferred with a Pasteur        pipette to a 15 mL conical screw-cap Falcon centrifuge tube.    -   PBS was added to bring the volume up to 14 mL and the tube was        mixed by inversion.    -   The tube was centrifuged at room temperature for 20-30 minutes        at 1300 rpm (approximately 320× g).    -   Immediately after centrifugation, as much supernatant as        possible was aspirated by vacuum and discarded without        disturbing the cell pellet.

4. Optional Wash

-   -   The cell pellet was re-suspended in the remaining liquid by        tapping against the bottom of the tube.    -   10 mL of PBS was added to the re-suspended cells, and        centrifuged at room temperature for 20 minutes at 1300 rpm.    -   Immediately after centrifugation, as much supernatant as        possible was aspirated by vacuum and discarded without        disturbing the cell pellet.

5. Optional: Freezing WBC Pellet

-   -   The cell pellet was mixed in the remaining liquid by tapping a        finger against the bottom of the tube.    -   0.5 to 1 mL of PBS was added to the re-suspended cells and one        half of the pellet was transferred (using a sterile tip on a        micropipette) to a labeled 1.8 mL cryovial.    -   The cryovial was centrifuged at room temperature for 5 minutes        at 5000 rpm (approximately 2250 g) in a microcentrifuge.    -   All of the supernatant liquid was discarded using a Pasteur        pipette without disturbing the cell pellet.    -   0.5 to 1 ml of RPMI 1640 containing 10% FBS and 5% DMSO was then        added to the tube and mixed a pipette and frozen overnight at        −80 C prior to transferring to a liquid nitrogen cell storage        freezer.        B. Establishment of T-Cell Cultures from Blood Specimens    -   1. The washed cells were re-suspended in 3.0 ml of RPMI 1640        medium with 10% Cosmic Calf Serum (CCS, Hyclone Laboratories,        Logan, Utah), about 25 ng/ml IL-2 (PreProTECH, Rocky Hill, N.J.)        and the manufacturer's recommended concentration of PHA (Life        Technology, Gaithersburg, Md.). The cells were then transferred        to an upright an upright 25 cm³ culture flask and incubated for        3-4 days at 37° C., 5% CO₂.    -   2. The cell culture was diluted to 5 ml with growth medium        (RPMI-1640, 10% FBS, 25 ng/ml IL-2). The cell concentration was        then adjusted to about 5×10⁵ cells/ml in the flask.    -   3. The growth of the cells was monitored daily. Cells were        maintained between 5×10⁵ and 1.5×10⁶ cells in an upright flask.        The depth of the medium in the flask did not exceed 1 cm (about        7 mLs in a T25 and 20 mLs in a T75). Cultures can be maintained        for approximately 21 days with a doubling time of about 24 hrs.        Senescence of the culture will be apparent by a dramatic        reduction in growth rate. Culture time may possibly be extended        by re-stimulation with PHA.    -   4. Optional-Freezing T-lymphocytes: T-lymphocytes may be frozen        at 3×10⁶ cells/vial using RPMI1640 medium containing 20% FCS and        7.5% DMSO. On day 5, 6, or 7 cryopreserve as many vials as        possible at 3×10⁶ cells/vial. This is sufficient to thaw 5 mLs        of culture at 5×10⁵ viable cells/ml.        When establishing T-cell cultures, the following should be        noted.    -   Fresh blood specimens should be collected in heparinized tubes        (or tubes containing an appropriate anti-coagulant) and used the        same day. ACD tubes should be used if specimens cannot be        processed within 24 hours. (Clin Chem 1988 January; 34(1):110-3;        Clin Diagn Lab Immunol. 1998 November; 5(6):804-7.).    -   Eight-10 mLs of blood is usually sufficient to establish 20        million cells by day 5.    -   T lymphocytes are the specific targets of the HIV virus. Use        extreme care if the HIV status of the patient is unknown.    -   Each new lot of IL-2 should be tested to determine the optimal        concentration. The lot from PreProTECH used for these        experiments was been found to be optimal at 25 ng/ml with only a        slight reduction in cell growth at concentrations up to 50        ng/ml.    -   Each lot of mitogen, e.g., phytohemagglutinin A (PHA), is        assayed by the supplier (Invitrogen) and should be used at the        recommended dilution.    -   All cultures are maintained in a water saturated atmosphere at        37 C, 5% CO₂.    -   Mononuclear cells and lymphocytes may also be collected using        either (lymphocyte separation medium (Ficoll-Hypaque) or        Lymphoprep tubes following the manufacturer's standard        procedure.

When analyzed by fluorescent activated cell sorting, the regimen of IL-2and PHA stimulation results in 99% CD3-positive cells (which stains allT cell subsets), with equal numbers of CD4-positive and CD4-negativecells (data not shown).

C. Chaperone Treatment

The density of the T cells was adjusted to 1×10⁶ per 3 ml of culturemedium (RPMI-1640, 10% FBS, 25 ng/ml IL-2). 3 ml (˜1×10⁶ cells) are thenpipetted into each of 6 wells of a labeled 6-well culture plate andincubated overnight at 37° C., 5% CO₂. 3 ml of additional medium wasthen added to 3 wells to give a final volume of 6 ml/well. To the threeremaining wells, 3 ml of medium containing DGJ (Cambridge MajorLaboratories, Inc., Germantown, Wis.) at a concentration of about 40 μM(2×; final concentration is 20 μM), for 4-5 days. Cells were harvestedby centrifugation (400×g for about 10 minutes) and washed 1× in 10 mlPBS. The resulting pellets were re-suspended in 1 ml PBS and transferredto a 1.7 ml microfuge tube and centrifuged in a refrigerated microfugeat 3000 rpm for 5 minutes. The supernatant was aspirated and the pelletswere stored frozen at −80° C. until assayed for enzyme activity.

Note that prior to conducting the enhancement assay, the optimumconcentration of DGJ was determined using a range from 2 nM-200 μM. Itwas determined that 20 μM was optimal.

D. Preparation of Fibroblasts

For a comparison, fibroblast cultures were prepared as describedpreviously (e.g., U.S. Pat. No. 6,274,597). Briefly, fibroblast cultureswere derived from skin biopsies of patients and grown in DMEM with 10%FBS until established (3-4 weeks).

E. Activity Assay

Prior to assay, the T cells were thawed on ice and sonicated for 2minutes, and all other assay reagents were thawed at room temperature.Fluorometric assay of α-GAL activity was performed essentially asdescribed previously (Kusiak et al., J Biol Chem. 1978; 253(1),184-190). The cells were lysed in 0.2 ml deionized water combined withvigorous pipetting and vortexing. The supernatant obtained aftercentrifugation at 13000 rpm for 2 min at 4° C. was put into a fresh tubeand used as the source of α-GAL. α-GAL activity was determined byincubating 50 μl aliquots of the supernatant (containing comparablequantities of protein as determined using 20 μl in a standard proteinquantitation assay) in a 24-well microplate at 37° C. with 3.75 mM4-methylumbelliferyl-α-D-galactopyranoside (4-MU Gal) (Research ProductsInternational, Mount Prospect, Ill.) in the citric acid/phosphate buffer(27 mM citrate/46 mM phosphate buffer pH 4.6) without taurocholate andwith BSA (3 mg/ml). The percentage of α-GAL was determined by comparingtotal activity with activity observed in the presence of 117 mMN-acetylgalactosamine (GalNAc) Sigma Chemical Co., St. Louis, Mo.), aspecific inhibitor of N-acetylgalactosaminidase. A Wallac 1420 Victor3™Fluorescence detection reader (Perkin Elmer, CA) was used to measure thereleased 4-MU at excitation and emission wavelengths of 355 nm and 460nm, respectively. Appropriate wells for fluorescent standards, andnegative (no substrate or no lysate) also were employed. For eachpatient sample at least three normal samples were tested concurrently.

Incubations were typically 30 minute duration but longer or shorterperiods may be employed with similar results.

Enzyme activity (nmol/hr/mg of protein) was calculated according to thefollowing:

$\frac{{Fluorescence}\mspace{14mu} {of}\mspace{14mu} {sample}}{{Fluorescence}\mspace{14mu} {of}\mspace{14mu} {Standard}}*\frac{60\mspace{14mu} {mins}}{{Incubation}\mspace{14mu} {time}\mspace{14mu} ({mins})}*\frac{1000\mspace{14mu} \mu \; L}{{Volume}\mspace{14mu} {assayed}\mspace{14mu} \left( {\mu \; L} \right)}*\frac{1}{{Protein}\mspace{14mu} {value}\mspace{14mu} \left( {{mg}/{mL}} \right)}$

One unit of enzyme activity is defined as the amount of enzyme thatcatalyzes the hydrolysis of 1 nmole of 4-methylumbelliferone per hour.The baseline “noise” in the fluorescence output was obtained byevaluating the average of blank six times. If the activity following SPCtreatment was at least 2 standard deviations above the baseline, it wasconsidered responsive and not noise.

For the comparative fibroblast enhancement assay, fibroblasts (˜1.5×10⁶)were grown in 12 ml culture medium in a T75 tissue culture flask in theabsence or presence of DGJ at 20 μM for 3 days. At the end of theincubation period, cells were removed from the flask by treatment withtrypsin-EDTA solution, collected by centrifugation and washed 3 timeswith phosphate-buffered saline. Cell pellets were frozen at −80 C untilassayed for α-GAL activity. All steps for processing the cell pellet forassay, including the extraction buffer, the time of sonication and thevolumes used are the same as used for the T-cells assayed above.

F. Western Blots

The level of α-GAL protein measured by Western blot. Protein wasdetermined using a Micro BCA Protein Assay kit (Pierce, Rockford, Ill.)with bovine serum albumin (BSA) as a standard. Absorbance at 562 nm wasmeasured using the Molecular Devices VersaMax absorbance reader in a96-well format. For gel electrophoresis prior to western blotting,proteins were separated using Novex Tris-glycine native or SDS-PAGE in8-16% gradient gels (Invitrogen). Western blots were developed usingrabbit polyclonal antibody against α-GAL was performed as describedpreviously (Park et al., Proc Natl Acad Sci USA. 2003; 100: 3450-54).

Results

This method described above using T cells is fast and effective whencompared with fibroblast-based α-GAL assays conducted substantiallysimilarly to the T cell assay (except that about 1.5×10⁶ fibroblastswere plated in each well instead of 2.5×10⁶ T cells).

Using this method, T cells from Fabry patients were incubated withoutand with 20 μM of DGJ for 1, 2, and 4 days, respectively and the α-GALactivity was measured in cell homogenates and compared to normal controlvalues. When the media was refreshed after 2 days and the cellsincubated for 2 additional days, α-GAL activity of A97V was 13% of thenormal control (FIG. 1, open circles). However, when 20 μM DGJ was addedto the medium of the T cells, the α-GAL activity increased to about 40%of normal after only 1 day of incubation and continued to 80% of normalafter 4 days of incubation (FIG. 1, filled circles). Addition of freshDGJ and media after 2 days and incubation for an additional 2 days didnot result in any change in the profile from that observed with a singleaddition of DGJ. The observed increase in activity after 3 days to alevel clearly distinguishable from the α-GAL activity without DGJ led tothe adoption of a standard time of measurement after 3 days ofincubation with 20 μM of DGJ in subsequent experiments. The use of athree day time course avoids the necessity to provide fresh media and/orsplitting the cells after 3 days in culture.

To determine the dosage effect of DGJ in T-cells from normal controls,α-GAL activity was measured in patient cells using a range of DGJ from 2nM to 200 μM (FIG. 2A) and compared to untreated normal control valuesassayed on the same day. α-GAL activity increased between 2 nM and 20 μMof DGJ. At 200 μM, DGJ inhibited normal α-GAL activity to approximately40% of the average of untreated normal controls. The optimal enhancementof mutated α-GAL activity within this same concentration range of DGJwas determined for the A97V mutation and compared with normal controls(FIG. 2B). Three separate experiments were carried out for the dosageeffects on A97V. DGJ in concentrations from 2 nM to 20 μM increased theα-GAL activity of A97V in a dose-dependent manner. However, at DGJconcentrations of 200 μM, there was a decrease in α-GAL activity whencompared to its highest level when cells were grown in 20 μM DGJ. In allthree experiments the optimal enhancement of activity of the A97Vmutation was observed at 20 μM DGJ with slightly lower activity at 2 and200 μM. When the mutations R112H and R301Q were tested in the sameconcentration range, a similar pattern emerged with the highest level ofenhanced activity observed at 20 μM DGJ (FIG. 2C). The results showedvarious mutations had similar dosage response profiles but differentlevels of enhancements. Among three α-GAL mutant genotypes tested forthe dosage effects, the 20 μM DGJ resulted in an increase in α-GAL to atleast 50% of the normal control.

The rescue effect of mutant α-GAL from patients with Fabry disease withat least 11 distinct genotypes has been observed using a pharmacologicalchaperone using this T cell-based α-GAL assay. Results, presented inTable 1, below, showed that DGJ enhanced the activity of at least fivedistinct mutant forms of α-GAL in T cells (T) and fibroblasts (F).However, the pharmacological chaperone did not enhance activity of fourdistinct mutant α-GAL forms. One classical Fabry patient's α-GALactivity was enhanced by DGJ at the intermediate level. The importanceof this assay lies in the fact that it can be used to screen forpatients who might benefit from pharmacological chaperoneadministration, thus avoiding the expense and frustration of unnecessarytherapy and tissue biopsies.

TABLE 1 Number of Activity Specimen Patient/ Replicates (% Normal)Enhancement Number Normal Sex Mutation (n =) (−DGJ) (+DGJ) ratio Group 1 PT M T41I 3 48 147 3.0 E  2 PT M T41I 4 61 175 2.9 E  3 PT M M51K 2 629 4.6 E  4 PT M A97V 3 14 75 5.5 E  5 PT M R112C 1 10 36 3.8 E  6 PT MR112C 3 8 49 6.3 E  7 PT M R112H 2 3 51 15.9 E  8 PT M R112H 2 8 73 9.4E  9 PT M R112H 3 3 60 20.0 E 10 PT M A143T 4 31 69 2.2 E 11 PT M A143T5 49 62 1.3 E 12 PT M S201F 1 9 82 9.6 E 13 PT M P205T 1 37 108 2.9 E 14PT M N215S 2 15 79 5.2 E 15 PT M P259R 1 9 297 32.6 E 16 PT M P259R 3 3138 47.4 E 17 PT M F295C 3 1 29 32.3 E 18 PT M L300P 5 2 72 36.1 E 19 PTM R301Q 1 7 91 12.3 E 20 PT M R301Q 3 22 204 9.1 E 21 PT M R301Q 4 7 8012.1 E 22 PT M G328A 4 2 54 24.6 E 23 PT M R49C 9 3 11 4.3 I 24 PT MY207S 3 4 15 3.5 I 25 PT M S276G 1 0 9 I 26 PT M S276G 3 1 12 9.8 I 27PT M C94S 2 2 2 NE NE 28 PT M G128E 1 2 4 NE NE 29 PT M G128E 5 2 2 NENE 30 PT M G132R 3 1 2 NE NE 31 PT M A143P 2 2 1 NE NE 32 PT M A143P 2 11 NE NE 33 PT M R220X 3 0 1 NE NE 34 PT M R227Q 3 4 3 NE NE 35 PT MW236R 3 1 2 NE NE 36 PT M G261D 1 0 1 NE NE 37 PT M G271C 1 2 3 NE NE 38PT M G271C 3 1 2 NE NE 39 PT M N272K 1 0 0 NE NE 40 PT M W287C 1 1 1 NENE 41 PT M W287C 1 1 2 NE NE 42 PT M R356W 1 1 1 NE NE 43 PT M R356W 4 00 NE NE 44 PT M dE358 3 2 4 NE NE 45 PT M L415P 1 0 3 NE NE 46 PT Munknown 1 1 2 NE NE 47 PT M unknown 2 0 1 NE NE 48 PT M 1042insG 3 1 1NE NE 49 PT M 256del1 1 0 0 NE NE 50 PT M 30delG 6 2 2 NE NE 51 PT M82insG 2 1 0 NE NE 52 PT M del26bp21 1 0 1 NE NE 53 PT M del26bp21 1 0 0NE NE 54 PT M ivs4-1g/a 2 0 0 NE NE 55 PT M Q119X 1 2 1 NE NE 56 PT MR220X 3 0 1 NE NE 57 PT M R301X 4 0 7 NE NE All NL NL M or F 162 101 1281.3 All NL NL M 115 101 124 1.2 Male All NL NL F 47 84 117 1.4 Female 58NL F 8 120 161 1.3 59 NL F 3 88 100 1.1 60 NL F 1 95 120 1.3 61 NL F 985 104 1.2 62 NL F 8 103 150 1.5 63 NL F 3 107 165 1.5 64 NL F 15 106145 1.4 65 NL M 8 100 97 1.0 66 NL M 4 80 141 1.8 67 NL M 1 172 198 1.168 NL M 1 271 315 1.2 69 NL M 1 110 140 1.3 70 NL M 1 101 145 1.4 71 NLM 19 106 138 1.3 72 NL M 11 90 131 1.4 73 NL M 4 75 99 1.3 74 NL M 10105 126 1.2 75 NL M 3 97 100 1.0 76 NL M 2 93 108 1.2 77 NL M 3 85 1161.4 78 NL M 3 89 118 1.3 79 NL M 1 127 107 0.8 80 NL M 42 100 117 1.2 81NL M 1 102 111 1.1 Abbreviations used: PT: patient; NL: normalindividual; E: enhanceable; I: enhanceable (intermediate); NE: notenhanceable;

It was determined the optimal enhancement of mutated α-GAL activity inthe in vitro assay was achieved using about 20 μM DGJ. Among three α-GALmutant genotypes tested for the dosage effects, the 20 μM DGJ resultedin an increase in α-GAL to at least 50% of the normal control for each(FIG. 3).

In Table 1, the enhanceable group included patients whose α-GAL activitywas at least 50% of normal controls when cultured in the presence of DGJ(e.g., R112H was enhanced to 60% of normal control activity). Includedin the enhanceable group are the mutations A97V, R301Q, R112H and L300P.For example, the activity of the A97V mutation increased from 14 to 75%of normal in the presence of DGJ, a 6-fold increase. Similarly R301Qincreased from 7 to 80% of normal with 12-fold, and R112H increased from3 to 60%, nearly a 20-fold change. In addition, activity in the L300Pmutation was increased from 2% to 72% of normal, a 37-fold increase,which was the highest among the enhanceable mutations examined. L300Pwas unusual in that some of the activity without DGJ was below theminimum threshold for detection. These results demonstratemutation-dependent enhancement levels and ratios.

The Western blots showed that the band intensity was considerablyincreased by treatment with DGJ in normal control cells and those withthe A97V and the R301Q mutation, while no increase was seen for R356W,G132R, and A143P (FIG. 3). The protein appears to have shifted to alower apparent molecular weight indicating maturation of the enzyme bypassage from the endoplasmic reticulum, through the Golgi apparatus tothe lysosome. The Western blots show that enhancement of α-GAL activityby DGJ is correlated with an increased amount of α-GAL protein. Aninstance where increased protein levels as measured by Western blots didnot result in higher enzyme activity has not yet been observed.

Discussion

The use of T cells in a test system for enhancement of enzymes by SPCsoffers significant advantages in the speed of assay and convenience overother culture systems. A critical step in determining which patients maybenefit from SPC therapy was the development of a rapid and reliablemethod for screening of patient-derived cells for enhancement of α-GALactivity by DGJ. The results demonstrate a method for quickly generatinga short-lived cell culture that permits the testing of the enhancementand also provides a useful system for future studies on the mechanism ofaction or for screening of additional chaperone molecules. Leukocytestraditionally used for the diagnosis of affected and carrier status donot survive long enough to permit repeat assays if necessary.

Although Epstein-Barr virus transformed B lymphoblasts (Fan et al., NatMed. 1999; 5(1), 112-115) and primary fibroblast cultures (Fan, supra;Mayes et al., Clin Chim Acta. 1981; 112(2), 247-251) have been tested,these are not convenient to use on a large scale for screening ofpatients for clinical studies. Primary fibroblast cultures require aninvasive skin punch biopsy and generally take at least three to fourweeks to grow enough cells for the assay. B cell lymphoblasts requireEpstein Barr viral transfection and selection process, which is time andlabor consuming, in addition to having unknown effects on enzymaticactivity.

The present invention provides a method for establishing T cell culturesfrom fresh blood of normal control individuals and patients with Fabrydisease. These cultures can be grown for use in an enhancement assay forα-GAL in 7 to 10 days. These data also show that the effectiveness ofDGJ enhancement was evident after about 3 days in the T cell growthmedia. The data generated are a reproducible measure of the degree ofenhanced enzyme activity by a SPC for a specific genotype.

This method can be used for other SPC-based enhancement assays of othergenetic diseases including glycosphingolipidoses, mucopolysaccharidoses,and glycogen storage disease (Pompe) and can be extended as a researchand clinical protocol in a wide range of genetically defined diseases,such as Cystic Fibrosis (CFTR) and cancer (p53, PTEN), and others.

Example 2: In Vivo Method for Evaluating Effects of an SPC on α-GALActivity

This example describes results from an open label Phase II study of DGJin Fabry patients (n=11) with 10 different α-GAL mutations and supportsthe use of the in vivo assay. The patients were selected for the PhaseII study based on the increase in α-GAL activity in the T-cell assaydescribed above. The genotypes were as follows: T41L (2 patients);A143T; A97V; M51K; S276G; L300P; G328A; P205T; N215S; and L415P.

Some patients (8) were administered DGJ according to the followingdosing schedule: 25 mg b.i.d. two weeks; 100 mg b.i.d. weeks 2-4; 250 mgb.i.d. weeks 4-6; and 25 mg b.i.d. weeks 6-12. Three patients received150 mg of DGJ every other day throughout the entire study. Blood wasdraw into an 8 mL Vacutainer CPT tube at the end of each dosing periodand treated as described below.

A. Preparation of Human WBC Pellets for Assay

WBCs were prepared substantially as described in Example 1, with theexception that no FBS/DMSO is added to the pellet prior to freezing.

The preliminary data is summarized in the following table.

B. Preparation of Human WBC Lysates for Assay

-   -   To the microtubes containing the WBC pellet, 0.6 ml of lysis        buffer (26 mM citrate/46 mM phosphate, pH 5.5) were added    -   Tubes were vortexed until the cells were re-suspended    -   Tubes were incubated at room temperature for about 15 minutes,        but agitate the suspension by vortexing every couple of minutes    -   Tubes were sonicated for 2 minutes, then vortexed for about 10        seconds    -   Lysates were incubated on ice until chilled, and then pooled        into a pre-chilled polyproylene container (on ice)    -   Container was vortexed and pooled lysates were divided into        0.100 mL aliquots in pre-chilled labeled 0.5 mL screw-cap        polypropylene microcentrifuge tubes. Pooled lysates were mixed        while aliquoting by vortexing between every 10-20 aliquots.    -   Aliquots were stored at −80° C. until use.

C. Human WBC Assay

-   -   Each tube containing lysate was thawed on ice, sonicated for 2        minutes, then vortexed for 1 minute.    -   50 μl of each standard, control, or clinical sample was added        into appropriate wells of a black polystyrene microplate (use 50        μl of 0.5% BSA in WBC lysis buffer for a standard)    -   50 μl of 117 mM GalNAc was added to all wells, and the plate was        incubated for 5 minutes at ambient temperature;    -   50 μl of 5 mM 4-MU Gal substrate was added to to all wells and        the wells were mixed on a plate shaker for 30 seconds    -   The plate was covered and incubated for about 1 hour at 37° C.    -   100 μl of 0.2M NaOH/Glycine buffer, pH 10.7 was added to each        well to stop the reaction    -   The plate was read using a fluorescent plate reader as described        in Example 1

Results

The available data from the first eleven patients treated with DGJ forat least 12 weeks suggest that treatment with DGJ leads to an increasein the activity of the enzyme deficient in Fabry disease in 10 of the 11patients (FIG. 4). The patient with the L415P genotype did not show anincrease following DGJ (at 6 weeks) (FIG. 5). For purposes ofcalculating the percentage of normal in the table, the level of α-GALthat is normal was derived by using the average of the levels of α-GALin white blood cells of 15 healthy volunteers from the multiple-dosePhase I study. The 11 patients represented 10 different geneticmutations and had baseline levels of α-GAL that ranged from zero to 30%of normal.

The data show an average 2-fold increase in α-GAL activity, and up to10-fold and 15-fold in some patients as measured in white blood cells.Activity remained elevated from 6-24 weeks and counting when the dosewas reduced back to 25 mg b.i.d (data not shown).

Discussion

Based on these results, it appeared possible to screen candidatepatients for eligibility for chaperone using an in vivo assay or an invitro assay using T cells as described in Example 1, since 10 out of 11patients who demonstrated a significant increase in α-GAL activity inthe T cell assay demonstrated an increase following a 2 week treatmentwith DGJ. Performing an in vivo screen may allow for a more accurateevaluation of the in vivo response to DGJ and other chaperone treatment,since the in vitro assay may not be fully predictive of an in vivoresponse due to systemic interactions, and may be especially useful todetermine appropriate dosing. This dramatic increase in activityappeared after 2 weeks at a low dose of only 50 mg/day (25 mg b.i.d.)(although different dosing regimens are contemplated by the presentinvention).

These methods can be used for chaperone based enhancement assays forother genetic diseases including glycosphingolipidoses,mucopolysaccharidoses and other lysosomal storage disorders in additionto other genetically based diseases, such as cystic fibrosis wherematuration of the protein occurs in the ER.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purpose.

1-34. (canceled)
 35. A method for treatment of Fabry disease in a humanpatient in need thereof, the method comprising administering to thepatient a therapeutically effective dose of 1-deoxygalactonojirimycin ora salt thereof every other day.